Growth-associated protease inhibitor heavy chain precursor

ABSTRACT

The invention provides a human growth-associated protease inhibitor heavy chain precursor (GAPIP) and polynucleotides which identify and encode GAPIP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of GAPIP.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/388,774 filed Sep. 2, 1999, which is a divisionalapplication of U.S. application Ser. No. 09/074,579 filed May 7, 1998,issued Dec. 14, 1999 as U.S. Pat. No. 6,001,596, all of whichapplications and patents are hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a growth-associated protease inhibitor heavy chain precursor and tothe use of these sequences in the diagnosis, treatment, and preventionof reproductive, developmental, neoplastic, and immunological disorders.

BACKGROUND OF THE INVENTION

[0003] Proteolytic processing is an essential component of normal cellgrowth, differentiation, remodeling, and homeostasis. The cleavage ofpeptide bonds within cells is necessary for the maturation of precursorproteins to their active form, the removal of signal sequences fromtargeted proteins, the degradation of incorrectly folded proteins, andthe controlled turnover of peptides within the cell. Proteasesparticipate in apoptosis, antigen presentation, inflammation, tissueremodeling during embryonic development, wound healing, and normalgrowth. They are necessary components of bacterial, parasitic, and viralinvasion and replication within a host. Four principal categories ofmammalian proteases have been identified based on active site structure,mechanism of action, and overall three-dimensional structure. (Beynon,R. J. and Bond, J. S. (1994) Proteolytic Enzymes: A Practical Approach,Oxford University Press, New York, N.Y., pp. 1-5.)

[0004] The serine proteases (SPs) are a large family of proteolyticenzymes that include the digestive enzymes, trypsin and chymotrypsin;components of the complement cascade and of the blood-clotting cascade;and enzymes that control the degradation and turnover of macromoleculesof the extracellular matrix. SPs are so named because of the presence ofa serine residue found in the active catalytic site for proteincleavage. The active site of all SP is composed of a triad of residuesincluding the aforementioned serine, an aspartate, and a histidineresidue. SPs have a wide range of substrate specificities and can besubdivided into subfamilies on the basis of these specificities. Themain sub-families are trypases which cleave after arginine or lysine;aspases which cleave after aspartate; chymases which cleave afterphenylalanine or leucine; metases which cleavage after methionine; andserases which cleave after serine.

[0005] The plasma inter-α-trypsin inhibitor family molecules are serineprotease inhibitors (serpins) composed of a 240 kDa plasma proteincomplex of at least five different types of glycoproteins. Theseglycoproteins consist of four heavy (H) chains and one 30 kDa light (L)chain named H1, H2, H3, H4, and L, and are independently synthesized andproteolytically processed from precursor proteins. (Daveau, M. et al.(1998) Arch. Biochem. Biophys. 350:315-323; and Salier, J. P. et al.(1992) Mamm. Genome 2:233-239.) The plasma inter-α-trypsin inhibitorlight chains have sequence similarity to the Kunitz trypsin inhibitorswhich appear to be present in all vertebrates (Salier, J. P. (1990)Trends Biochem. Sci. 15:435439.) Some examples of the Kunitz trypsininhibitors are tissue factor pathway inhibitor, which regulates tissuefactor-induced coagulation, and protease nexin-2, which regulates serumcoagulation factor XIa. (Broze, G. J. (1995) Annu. Rev. Med. 46:103-112;and Wagner, S. L. et al. (1993) Brain Res. 626:90-98.) The heavy chainprecursors encode a signal peptide sequence and the mature chain. Otherplasma inter-α-trypsin inhibitor heavy chains have been described inhuman and rodents. (Bourguignon, J. et al. (1993) Eur. J. Biochem.212:771-776; Salier, 1992, supra; and Salier, J. P. (1996) Biochem. J.315:1-9.) Proteases and protease inhibitory molecules may contain aminoacid sequence motifs which determine protein-protein interactions, suchas the potential metal-binding site of von Willebrand factor type A3(vWFA3) motif, glycine-amino acid-serine-amino acid-serine. This motifis also required for ligand interaction in the homologous I-type domainsof integrins CR3 and LFA-1. (Huizinga, E. G. (1997) Structure5:1147-1156.)

[0006] The expression of the rat plasma inter-α-trypsin inhibitor genesis regulated by inflammation in vivo. The genes are predominantlyexpressed in the rat liver, but H2 and H3 mRNA is also present in brain,intestine, and stomach. (Daveau, supra.)

[0007] Protease inhibitors play a major role in the regulation of theactivity and effect of proteases. They have been shown to controlpathogenesis in animal models of proteolytic disorders and in thetreatment of HIV. (Murphy, G. (1991) Agents Actions Suppl. 35:69-76; andPakyz, A. and Isreal, D. (1997) J. Am. Pharm. Assoc. (Wash.)NS37:543-551.)

[0008] The discovery of a new growth-associated protease inhibitor heavychain precursor and the polynucleotides encoding it satisfies a need inthe art by providing new compositions which are useful in the diagnosis,treatment, and prevention of reproductive, developmental, neoplastic,and immunological disorders.

SUMMARY OF THE INVENTION

[0009] The invention is based on the discovery of a new humangrowth-associated protease inhibitor heavy chain precursor (GAPIP), thepolynucleotides encoding GAPIP, and the use of these compositions forthe diagnosis, treatment, or prevention of reproductive, developmental,neoplastic, and immunological disorders.

[0010] The invention features a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

[0011] The invention further provides a substantially purified varianthaving at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoprovides an isolated and purified polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. The invention also includes an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide sequenceidentity to the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0012] The invention further provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0013] The invention also provides an isolated and purifiedpolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2, and an isolated and purified polynucleotidevariant having at least 90% polynucleotide sequence identity to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides an isolated andpurified polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2.

[0014] The invention further provides an expression vector comprising atleast a fragment of the polynucleotide encoding the polypeptidecomprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Inanother aspect, the expression vector is contained within a host cell.

[0015] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcomprising an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

[0016] The invention also provides a pharmaceutical compositioncomprising a substantially purified polypeptide having the sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

[0017] The invention further includes a purified antibody which binds toa polypeptide comprising the sequence of SEQ ID NO:1 or a fragment ofSEQ ID NO:1, as well as a purified agonist and a purified antagonist ofthe polypeptide.

[0018] The invention also provides a method for treating or preventing areproductive disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0019] The invention also provides a method for treating or preventing adevelopmental disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0020] The invention also provides a method for treating or preventing aneoplastic disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of thepolypeptide having the amino acid sequence of SEQ ID NO:1 or a fragmentof SEQ ID NO:1.

[0021] The invention also provides a method for treating or preventingan immunological disorder, the method comprising administering to asubject in need of such treatment an effective amount of an antagonistof the polypeptide having the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

[0022] The invention also provides a method for detecting apolynucleotide encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological samplecontaining nucleic acids, the method comprising the steps of: (a)hybridizing the complement of the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1 to at least one of the nucleic acids of thebiological sample, thereby forming a hybridization complex; and (b)detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1 in the biological sample. In oneaspect, the nucleic acids of the biological sample are amplified by thepolymerase chain reaction prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J show the aminoacid sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID NO:2) ofGAPIP. The alignment was produced using MacDNASIS PRO software (HitachiSoftware Engineering Co. Ltd., San Bruno, Calif.).

[0024]FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show the amino acid sequencealignments among GAPIP (688183; SEQ ID NO:1), human pre-inter-α-trypsininhibitor (GI 33985; SEQ ID NO:3), human pre-inter-α-trypsin inhibitorheavy chain H1 (GI 33989; SEQ ID NO:4), and human pre-inter-α-trypsininhibitor heavy chain H3 (GI 288563; SEQ ID NO:5), produced using themultisequence alignment program of LASERGENE software (DNASTAR Inc,Madison Wis.).

[0025]FIG. 3 shows the amino acid sequence phylogenic tree among GAPIP(688183; SEQ ID NO:1), human pre-inter-α-trypsin inhibitor (GI 33985;SEQ ID NO:3), human pre-inter-α-trypsin inhibitor heavy chain H1 (GI33989; SEQ ID NO:4), and human pre-inter-α-trypsin inhibitor heavy chainH3 (GI 288563; SEQ ID NO:5), produced using the multisequence alignmentprogram of LASERGENE software (DNASTAR Inc).

DESCRIPTION OF THE INVENTION

[0026] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0027] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0028] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs.

[0029] Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods, devices, and materials are nowdescribed. All publications mentioned herein are cited for the purposeof describing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications and which might be used inconnection with the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

[0030] Definitions

[0031] “GAPIP,” as used herein, refers to the amino acid sequences ofsubstantially purified GAPIP obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0032] The term “agonist,” as used herein, refers to a molecule which,when bound to GAPIP, increases or prolongs the duration of the effect ofGAPIP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of GAPIP.

[0033] An “allelic variant,” as this term is used herein, is analternative form of the gene encoding GAPIP. Allelic variants may resultfrom at least one mutation in the nucleic acid sequence and may resultin altered mRNAs or in polypeptides whose structure or function may ormay not be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

[0034] “Altered” nucleic acid sequences encoding GAPIP, as describedherein, include those sequences with deletions, insertions, orsubstitutions of different nucleotides, resulting in a polynucleotidethe same as GAPIP or a polypeptide with at least one functionalcharacteristic of GAPIP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding GAPIP,and improper or unexpected hybridization to allelic variants, with alocus other than the normal chromosomal locus for the polynucleotidesequence encoding GAPIP. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent GAPIP. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of GAPIP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

[0035] The terms “amino acid” or “amino acid sequence,” as used herein,refer to an oligopeptide, peptide, polypeptide, or protein sequence, ora fragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of GAPIP which are preferablyabout 5 to about 15 amino acids in length, most preferably 14 aminoacids, and which retain some biological activity or immunologicalactivity of GAPIP. Where “amino acid sequence” is recited herein torefer to an amino acid sequence of a naturally occurring proteinmolecule, “amino acid sequence” and like terms are not meant to limitthe amino acid sequence to the complete native amino acid sequenceassociated with the recited protein molecule.

[0036] “Amplification,” as used herein, relates to the production ofadditional copies of a nucleic acid sequence. Amplification is generallycarried out using polymerase chain reaction (PCR) technologies wellknown in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler(1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., pp. 1-5.)

[0037] The term “antagonist,” as it is used herein, refers to a moleculewhich, when bound to GAPIP, decreases the amount or the duration of theeffect of the biological or immunological activity of GAPIP. Antagonistsmay include proteins, nucleic acids, carbohydrates, antibodies, or anyother molecules which decrease the effect of GAPIP.

[0038] As used herein, the term “antibody” refers to intact molecules aswell as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments,which are capable of binding the epitopic determinant.

[0039] Antibodies that bind GAPIP polypeptides can be prepared usingintact polypeptides or using fragments containing small peptides ofinterest as the immunizing antigen. The polypeptide or oligopeptide usedto immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derivedfrom the translation of RNA, or synthesized chemically, and can beconjugated to a carrier protein if desired.

[0040] Commonly used carriers that are chemically coupled to peptidesinclude bovine serum albumin, thyroglobulin, and keyhole limpethemocyanin (KLH). The coupled peptide is then used to immunize theanimal. The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

[0041] The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

[0042] As used herein, the term “biologically active,” refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic GAPIP, or ofany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0043] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

[0044] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding GAPIP orfragments of GAPIP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g., sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

[0045] “Consensus sequence,” as used herein, refers to a nucleic acidsequence which has been resequenced to resolve uncalled bases, extendedusing XL-PCR (Applied Biosystems, Foster City Calif.) in the 5′ and/orthe 3′ direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW Fragment Assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

[0046] As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding GAPIP, byNorthern analysis is indicative of the presence of nucleic acidsencoding GAPIP in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding GAPIP.

[0047] A “deletion,” as the term is used herein, refers to a change inthe amino acid or nucleotide sequence that results in the absence of oneor more amino acid residues or nucleotides.

[0048] The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0049] The term “similarity,” as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word “identity” may substitute for the word “similarity.” Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

[0050] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, Inc.). TheMEGALIGN program can create alignments between two or more sequencesaccording to different methods, e.g., the clustal method. (See, e.g.,Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustalalgorithm groups sequences into clusters by examining the distancesbetween all pairs. The clusters are aligned pairwise and then in groups.The percentage similarity between two amino acid sequences, e.g.,sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

[0051] “Human artificial chromosomes” (HACs), as described herein, arelinear microchromosomes which may contain DNA sequences of about 6 kb to10 Mb in size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

[0052] The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

[0053] “Hybridization,” as the term is used herein, refers to anyprocess by which a strand of nucleic acid binds with a complementarystrand through base pairing.

[0054] As used herein, the term “hybridization complex” refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀t or R₀t analysis) or formedbetween one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0055] The words “insertion” or “addition,” as used herein, refer tochanges in an amino acid or nucleotide sequence resulting in theaddition of one or more amino acid residues or nucleotides,respectively, to the sequence found in the naturally occurring molecule.

[0056] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0057] The term “microarray,” as used herein, refers to an arrangementof distinct polynucleotides arrayed on a substrate, e.g., paper, nylonor any other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

[0058] The terms “element” or “array element” as used herein in amicroarray context, refer to hybridizable polynucleotides arranged onthe surface of a substrate.

[0059] The term “modulate,” as it appears herein, refers to a change inthe activity of GAPIP. For example, modulation may cause an increase ora decrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of GAPIP.

[0060] The phrases “nucleic acid” or “nucleic acid sequence,” as usedherein, refer to a nucleotide, oligonucleotide, polynucleotide, or anyfragment thereof. These phrases also refer to DNA or RNA of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent the sense or the antisense strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material. In this context,“fragments” refers to those nucleic acid sequences which, whentranslated, would produce polypeptides retaining some functionalcharacteristic, e.g., antigenicity, or structural domain characteristic,e.g., ATP-binding site, of the full-length polypeptide.

[0061] The terms “operably associated” or “operably linked,” as usedherein, refer to functionally related nucleic acid sequences. A promoteris operably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

[0062] The term “oligonucleotide,” as used herein, refers to a nucleicacid sequence of at least about 6 nucleotides to 60 nucleotides,preferably about 15 to 30 nucleotides, and most preferably about 20 to25 nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

[0063] “Peptide nucleic acid” (PNA), as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least about 5 nucleotides in length linked to a peptide backboneof amino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

[0064] The term “sample,” as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acids encodingGAPIP, or fragments thereof, or GAPIP itself, may comprise a bodilyfluid; an extract from a cell, chromosome, organelle, or membraneisolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution orbound to a solid support; a tissue; a tissue print; etc.

[0065] As used herein, the terms “specific binding” or “specificallybinding” refer to that interaction between a protein or peptide and anagonist, an antibody, or an antagonist. The interaction is dependentupon the presence of a particular structure of the protein, e.g., theantigenic determinant or epitope, recognized by the binding molecule.For example, if an antibody is specific for epitope “A,” the presence ofa polypeptide containing the epitope A, or the presence of freeunlabeled A, in a reaction containing free labeled A and the antibodywill reduce the amount of labeled A that binds to the antibody.

[0066] As used herein, the term “stringent conditions” refers toconditions which permit hybridization between polynucleotides and theclaimed polynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0067] For example, stringent salt concentration will ordinarily be lessthan about 750 MM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

[0068] The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0069] The term “substantially purified,” as used herein, refers tonucleic acid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

[0070] A “substitution,” as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0071] “Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

[0072] A “variant” of GAPIP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software.

[0073] The Invention

[0074] The invention is based on the discovery of a new humangrowth-associated protease inhibitor heavy chain precursor (GAPIP), thepolynucleotides encoding GAPIP, and the use of these compositions forthe diagnosis, treatment, or prevention of reproductive, developmental,neoplastic, and immunological disorders.

[0075] Nucleic acids encoding the GAPIP of the present invention werefirst identified in Incyte Clone 688183 from the uterus cDNA library(UTRSNOT02) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 688183 (UTRSNOT02), 3043969 (HEAANOT01), 3112673 (BRSTNOT17),3052595 (LNODNOTO8), 789100 (PROSTUT03), 785182 (PROSNOT05), 1505061 and1505717 (BRAITUT07), 1794195 and 1795083 (PROSTUT05), 2125590(BRSTNOT07), 1558218 (SPLNNOT04), 1361072 (LUNGNOT12), and 1964439(BRSTNOT04).

[0076] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J. GAPIP is 942 amino acids inlength and has eight potential N-glycosylation sites at residues N97,N127, N231, N421, N508, N776, N795, and N862; twelve potential caseinkinase II phosphorylation sites at residues S17, S28, T112, T129, S158,S269, S354, T410, T581, S592, T676, and S754; two potentialglycosaminoglycan attachment sites at residues S213 and S391; seventeenpotential protein kinase C phosphorylation sites at residues S55, S70,T112, S175, S182, S213, S337, S354, T416, T458, S535, S559, T581, S611,S620, S651, and T880; one potential tyrosine kinase phosphorylation siteat residue Y919; a potential signal peptide sequence from M1 to C14; anda vWFA3 domain, which contains the potential metal-binding siteglycine-amino acid-serine-amino acid-serine, from N295 to N440. As shownin FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G, GAPIP has chemical andstructural similarity with human pre-inter-α-trypsin inhibitor (GI33985; SEQ ID NO:3), human pre-inter-α-trypsin inhibitor heavy chain H1(GI 33989; SEQ ID NO:4), and human pre-inter-α-trypsin inhibitor heavychain H3 (GI 288563; SEQ ID NO:5). In particular, GAPIP and humanpre-inter-α-trypsin inhibitor share 28% identity, one potentialN-glycosylation site, four potential casein kinase II phosphorylationsites, four potential protein kinase C phosphorylation sites, thepotential signal peptide sequence, and the vWFA3 potential metal-bindingsite glycine-amino acid-serine-amino acid-serine. In addition, GAPIP andhuman pre-inter-α-trypsin inhibitor heavy chains H1 and H3 share 27% and23% identity, respectively, one potential N-glycosylation site, fourpotential casein kinase II phosphorylation sites, five potential proteinkinase C phosphorylation sites, the potential signal peptide sequence,and the vWFA3 potential metal-binding site glycine-aminoacid-serine-amino acid-serine. As illustrated by FIG. 3, GAPIP and humanpre-inter-α-trypsin inhibitor heavy chains share a common phylogenicheritage. A fragment of SEQ ID NO:2 from about nucleotide 982 to aboutnucleotide 1011 is useful, for example, for designing oligonucleotidesor as a hybridization probe. Northern analysis shows the expression ofthis sequence in various libraries, at least 63% of which areimmortalized or cancerous and at least 26% of which involve immuneresponse. Of particular note is the expression of GAPIP in reproductive,gastrointestinal, nervous, and fetal tissues.

[0077] The invention also encompasses GAPIP variants. A preferred GAPIPvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the GAPIP amino acid sequence, and which contains at leastone functional or structural characteristic of GAPIP.

[0078] The invention also encompasses polynucleotides which encodeGAPIP. In a particular embodiment, the invention encompasses apolynucleotide sequence comprising the sequence of SEQ ID NO:2, whichencodes an GAPIP.

[0079] The invention also encompasses a variant of a polynucleotidesequence encoding GAPIP. In particular, such a variant polynucleotidesequence will have at least about 80%, more preferably at least about90%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding GAPIP. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of GAPIP.

[0080] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding GAPIP, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringGAPIP, and all such variations are to be considered as beingspecifically disclosed.

[0081] Although nucleotide sequences which encode GAPIP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring GAPIP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding GAPIP or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding GAPIP and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0082] The invention also encompasses production of DNA sequences whichencode GAPIP and GAPIP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingGAPIP or any fragment thereof.

[0083] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:2, or a fragment of SEQID NO:2, under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.)

[0084] Methods for DNA sequencing are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNApolymerase (Amersham Pharmacia Biotech (APB), Piscataway N.J.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 system (Hamilton, Reno Nev.) and theDNA ENGINE thermal cycler (MJ Research, Watertown Mass.). Machinescommonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNAsequencing systems (Applied Biosystems), the MEGABACE 1000 DNAsequencing system (APB), and the like.

[0085] The nucleic acid sequences encoding GAPIP may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer Analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

[0086] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0087] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Applied Biosystems), and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0088] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode GAPIP may be cloned in recombinant DNAmolecules that direct expression of GAPIP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express GAPIP.

[0089] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterGAPIP-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0090] In another embodiment, sequences encoding GAPIP may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp.Ser. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids Symp. Ser.7:225-232.) Alternatively, GAPIP itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (Perkin Elmer).Additionally, the amino acid sequence of GAPIP, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0091] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

[0092] In order to express a biologically active GAPIP, the nucleotidesequences encoding GAPIP or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding GAPIP. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding GAPIP. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding GAPIP and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0093] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding GAPIPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

[0094] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding GAPIP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0095] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding GAPIP. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding GAPIP can be achievedusing a multifunctional E. coli vector such as BLUESCRIPT phagemid(Stratagene, La Jolla, Calif.) or PSPORT 1 plasmid (Life Technologies).Ligation of sequences encoding GAPIP into the vector's multiple cloningsite disrupts the lacZ gene, allowing a calorimetric screening procedurefor identification of transformed bacteria containing recombinantmolecules. In addition, these vectors may be useful for in vitrotranscription, dideoxy sequencing, single strand rescue with helperphage, and creation of nested deletions in the cloned sequence. (See,e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509.) When large quantities of GAPIP are needed, e.g. for theproduction of antibodies, vectors which direct high level expression ofGAPIP may be used. For example, vectors containing the strong, inducibleT5 or T7 bacteriophage promoter may be used.

[0096] Yeast expression systems may be used for production of GAPIP. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-54; Scorer, C. A. et al. (1994)Bio/Technology 12:181-184.)

[0097] Plant systems may also be used for expression of GAPIP.Transcription of sequences encoding GAPIP may be driven viral promoters,e.g., the 35S and 19S promoters of CaMV used alone or in combinationwith the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.6:307-311.) Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. etal. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.17:85-105.) These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. (See, e.g.,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.) Inmammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding GAPIP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses GAPIP in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0098] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes.

[0099] For long term production of recombinant proteins in mammaliansystems, stable expression of GAPIP in cell lines is preferred. Forexample, sequences encoding GAPIP can be transformed into cell linesusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for about 1 to 2 days in enrichedmedia before being switched to selective media. The purpose of theselectable marker is to confer resistance to a selective agent, and itspresence allows growth and recovery of cells which successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be propagated using tissue culture techniques appropriate to thecell type.

[0100] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; andLowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides neomycin and G418; and als orpat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14; and Murry, supra.) Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto,Calif.), β glucuronidase and its substrate β-D-glucuronoside, orluciferase and its substrate luciferin may be used. These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55:121-131.) Although the presence/absence of marker geneexpression suggests that the gene of interest is also present, thepresence and expression of the gene may need to be confirmed. Forexample, if the sequence encoding GAPIP is inserted within a marker genesequence, transformed cells containing sequences encoding GAPIP can beidentified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with a sequence encoding GAPIP underthe control of a single promoter. Expression of the marker gene inresponse to induction or selection usually indicates expression of thetandem gene as well.

[0101] In general, host cells that contain the nucleic acid sequenceencoding GAPIP and that express GAPIP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0102] Immunological methods for detecting and measuring the expressionof GAPIP using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on GAPIP is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E.et al. (1997 and periodic supplements) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; andMaddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

[0103] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding GAPIPinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding GAPIP, or any fragments thereof, may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byPromega (Madison Wis.) or Amersham Pharmacia Biotech. Suitable reportermolecules or labels which may be used for ease of detection includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents, as well as substrates, cofactors, inhibitors, magneticparticles, and the like.

[0104] Host cells transformed with nucleotide sequences encoding GAPIPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode GAPIP may be designed to contain signal sequences which directsecretion of GAPIP through a prokaryotic or eukaryotic cell membrane.

[0105] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, BEK293, and W138), are available from the American TypeCulture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0106] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding GAPIP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric GAPIPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of GAPIP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the GAPIP encodingsequence and the heterologous protein sequence, so that GAPIP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

[0107] In a further embodiment of the invention, synthesis ofradiolabeled GAPIP may be achieved in vitro using the TNT rabbitreticulocyte lysate or wheat germ extract systems (Promega). Thesesystems couple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

[0108] Fragments of GAPIP may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the Applied Biosystems431A peptide synthesizer. Various fragments of GAPIP may be synthesizedseparately and then combined to produce the full length molecule.

[0109] Therapeutics

[0110] Chemical and structural similarity exists among GAPIP and humanpre-inter-α-trypsin inhibitor (GI 33985; SEQ ID NO:3), humanpre-inter-α-trypsin inhibitor heavy chain H1 (GI 33989; SEQ ID NO:4),and human pre-inter-α-trypsin inhibitor heavy chain H3 (GI 288563; SEQID NO:5). In addition, GAPIP is expressed in cancer, immune,reproductive, gastrointestinal, nervous, and fetal tissues. Therefore,GAPIP appears to play a role in reproductive, developmental, neoplastic,and immunological disorders.

[0111] Therefore, in one embodiment, a pharmaceutical compositioncomprising a substantially purified GAPIP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a reproductive disorder. Such reproductive disorders caninclude, but are not limited to, disorders of prolactin production;infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, uterine fibroids, autoimmunedisorders, ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, carcinoma of the male breast, and gynecomastia.

[0112] In another embodiment, a vector capable of expressing GAPIP or afragment or derivative thereof may be administered to a subject to treator prevent a reproductive disorder including, but not limited to, thosedescribed above.

[0113] In a further embodiment, GAPIP or a fragment or derivativethereof may be administered to a subject to treat or prevent areproductive disorder including, but not limited to, those providedabove.

[0114] In still another embodiment, an agonist which modulates theactivity of GAPIP may be administered to a subject to treat or prevent areproductive disorder including, but not limited to, those listed above.

[0115] In one embodiment, a pharmaceutical composition comprising asubstantially purified GAPIP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a developmental disorder. The term “developmental disorder”refers to any disorder associated with growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject (such as the brain, adrenal gland, kidney, skeletal orreproductive system). Such developmental disorders can include, but arenot limited to, renal tubular acidosis, anemia, Cushing's syndrome,achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia,genitourinary abnormalities, and mental retardation), Smith-Magenissyndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spinal bifida, congenital glaucoma, cataract, and sensorineuralhearing loss.

[0116] In another embodiment, a vector capable of expressing GAPIP or afragment or derivative thereof may be administered to a subject to treator prevent a developmental disorder including, but not limited to, thosedescribed above.

[0117] In a further embodiment, GAPIP or a fragment or derivativethereof may be administered to a subject to treat or prevent adevelopmental disorder including, but not limited to, those providedabove.

[0118] In still another embodiment, an agonist which modulates theactivity of GAPIP may be administered to a subject to treat or prevent adevelopmental disorder including, but not limited to, those listedabove.

[0119] In one embodiment, an antagonist of GAPIP may be administered toa subject to treat or prevent a neoplastic disorder. Such neoplasticdisorders may include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, an antibody which specifically binds GAPIP may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress GAPIP.

[0120] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding GAPIP may be administered to a subject totreat or prevent a neoplastic disorder including, but not limited to,those described above.

[0121] In one embodiment, an antagonist of GAPIP may be administered toa subject to treat or prevent an immunological disorder. Suchimmunological disorders may include, but are not limited to, acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; and arteriosclerosis, bursitis,cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,and primary thrombocythemia. In one aspect, an antibody whichspecifically binds GAPIP may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express GAPIP.

[0122] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding GAPIP may be administered to a subject totreat or prevent an immunological disorder including, but not limitedto, those described above. In other embodiments, any of the proteins,antagonists, antibodies, agonists, complementary sequences, or vectorsof the invention may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0123] An antagonist of GAPIP may be produced using methods which aregenerally known in the art. In particular, purified GAPIP may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind GAPIP. Antibodies to GAPIP mayalso be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0124] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith GAPIP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0125] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to GAPIP have an amino acid sequenceconsisting of at least about 5 amino acids, and, more preferably, of atleast about 10 amino acids. It is also preferable that theseoligopeptides, peptides, or fragments are identical to a portion of theamino acid sequence of the natural protein and contain the entire aminoacid sequence of a small, naturally occurring molecule. Short stretchesof GAPIP amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0126] Monoclonal antibodies to GAPIP may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0127] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce GAPIP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

[0128] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

[0129] Antibody fragments which contain specific binding sites for GAPIPmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

[0130] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between GAPIP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering GAPIP epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

[0131] In another embodiment of the invention, the polynucleotidesencoding GAPIP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding GAPIP may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding GAPIP. Thus, complementary molecules orfragments may be used to modulate GAPIP activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding GAPIP.

[0132] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding GAPIP. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

[0133] Genes encoding GAPIP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding GAPIP. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0134] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding GAPIP. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0135] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingGAPIP.

[0136] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0137] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding GAPIP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0138] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0139] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15:462466.) Any of the therapeuticmethods described above may be applied to any subject in need of suchtherapy, including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

[0140] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of GAPIP, antibodies to GAPIP, and mimetics, agonists,antagonists, or inhibitors of GAPIP. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0141] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0142] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0143] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0144] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0145] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0146] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0147] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0148] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0149] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0150] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0151] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of GAPIP, such labeling wouldinclude amount, frequency, and method of administration.

[0152] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0153] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0154] A therapeutically effective dose refers to that amount of activeingredient, for example GAPIP or fragments thereof, antibodies of GAPIP,and agonists, antagonists or inhibitors of GAPIP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0155] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0156] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0157] Diagnostics

[0158] In another embodiment, antibodies which specifically bind GAPIPmay be used for the diagnosis of disorders characterized by expressionof GAPIP, or in assays to monitor patients being treated with GAPIP oragonists, antagonists, or inhibitors of GAPIP. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for GAPIP include methodswhich utilize the antibody and a label to detect GAPIP in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

[0159] A variety of protocols for measuring GAPIP, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of GAPIP expression. Normal or standardvalues for GAPIP expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to GAPIP under conditions suitable for complex formation.The amount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of GAPIP expressedin subject samples, control and disease, from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0160] In another embodiment of the invention, the polynucleotidesencoding GAPIP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof GAPIP may be correlated with disease. The diagnostic assay may beused to determine absence, presence, and excess expression of GAPIP, andto monitor regulation of GAPIP levels during therapeutic intervention.

[0161] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding GAPIP or closely related molecules may be used to identifynucleic acid sequences which encode GAPIP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding GAPIP, allelicvariants, or related sequences.

[0162] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of theGAPIP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the GAPIP gene.

[0163] Means for producing specific hybridization probes for DNAsencoding GAPIP include the cloning of polynucleotide sequences encodingGAPIP or GAPIP derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²P or 35S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0164] Polynucleotide sequences encoding GAPIP may be used for thediagnosis of a disorder associated with expression of GAPIP. Examples ofsuch a disorder include, but are not limited to, a reproductivedisorder, such as, disorders of prolactin production; infertility,including tubal disease, ovulatory defects, and endometriosis;disruptions of the estrous cycle, disruptions of the menstrual cycle,polycystic ovary syndrome, ovarian hyperstimulation syndrome,endometrial and ovarian tumors, uterine fibroids, autoimmune disorders,ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, carcinoma of the male breast, and gynecomastia; a developmentaldisorder, such as, renal tubular acidosis, anemia, Cushing's syndrome,achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia,genitourinary abnormalities, and mental retardation), Smith-Magenissyndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spinal bifida, congenital glaucoma, cataract, and sensorineuralhearing loss; a neoplastic disorder, such as, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; and an immunological disorder, such as, acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; and arteriosclerosis, bursitis,cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,and primary thrombocythemia. The polynucleotide sequences encoding GAPIPmay be used in Southern or Northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; in dipstick, pin, andELISA assays; and in microarrays utilizing fluids or tissues frompatients to detect altered GAPIP expression. Such qualitative orquantitative methods are well known in the art.

[0165] In a particular aspect, the nucleotide sequences encoding GAPIPmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding GAPIP may be labeled by standard methods and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding GAPIP in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0166] In order to provide a basis for the diagnosis of a disorderassociated with expression of GAPIP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding GAPIP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0167] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0168] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0169] Additional diagnostic uses for oligonucleotides designed from thesequences encoding GAPIP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding GAPIP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding GAPIP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0170] Methods which may also be used to quantitate the expression ofGAPIP include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and interpolating resultsfrom standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol.Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem.212:229-236.) The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0171] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0172] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0173] In another embodiment of the invention, nucleic acid sequencesencoding GAPIP may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev.7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)

[0174] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) MolecularBiology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding GAPIP on a physicalchromosomal map and a specific disorder, or a predisposition to aspecific disorder, may help define the region of DNA associated withthat disorder. The nucleotide sequences of the invention may be used todetect differences in gene sequences among normal, carrier, and affectedindividuals.

[0175] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336:577-580.) The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0176] In another embodiment of the invention, GAPIP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenGAPIP and the agent being tested may be measured.

[0177] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with GAPIP, orfragments thereof, and washed. Bound GAPIP is then detected by methodswell known in the art. Purified GAPIP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0178] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding GAPIPspecifically compete with a test compound for binding GAPIP. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with GAPIP.

[0179] In additional embodiments, the nucleotide sequences which encodeGAPIP may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0180] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0181] I. cDNA Library Construction

[0182] The UTRSNOT02 cDNA library was constructed from uterus tissueobtained from a 34-year old Caucasian female (specimen #0047A) during avaginal hysterectomy. Pathology indicated no diagnostic abnormality inthe uterus or cervix. However, the left ovarian tissue showed dilatedfollicular cystis, all embedded. Patient history included the diagnosesof dysmenorrhea, dyspareunia, hemorrhoids and alcohol use. The patientwas not taking any medications. Family history included malignantstomach neoplasm in the mother; benign large bowel neoplasm in thefather; congenital heart anomaly, irritable bowel syndrome, andulcerative colitis in the siblings; colon cancer in the paternal aunt atage sixty and colon cancer in paternal uncle at age eleven; andcerebrovascular disease, type II diabetes, and depression in thegrandparents.

[0183] The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an SW28 rotor in an L8-70M ultracentrifuge(Beckman Coulter, Fullerton Calif.) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. RNAextraction and precipitation were repeated as before. The mRNA was thenisolated using the QIAGEN OLIGOTEX kit (QIAGEN Inc., Chatsworth Calif.)and used to construct the cDNA library.

[0184] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system (Life Technologies). cDNA synthesis wasinitiated with a NotI-oligo d(T) primer.

[0185] Double-stranded cDNA was blunted, ligated to SalI adaptors,digested with NotI, fractionated on a SEPHAROSE CL4B column (Catalog#275105-01, APB), and those cDNAs exceeding 400 bp were into the NotIand SalI sites of the PSPORT 1 vector. The plasmid PSPORT 1 wassubsequently transformed into DH5α competent cells (Catalog #18258-012,Life Technologies).

[0186] II Isolation and Sequencing of cDNA Clones

[0187] Plasmid DNA was released from the cells and purified using theREAL PREP 96 plasmid kit (QIAGEN Inc.). The recommended protocol wasemployed except for the following changes: 1) the bacteria were culturedin 1 ml of sterile TERRIFIC BROTH (Life Technologies) with carbenicillinat 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0188] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno,Nev.) in combination with the DNA ENGINE thermal cyclers (MJ Research)and Applied Biosystems 377 DNA Sequencing Systems; and the reading framewas determined.

[0189] III. Similarity Searching of cDNA Clones and Their DeducedProteins

[0190] The nucleotide sequences and/or amino acid sequences of theSequence Listing were used to query sequences in the GenBank, SwissProt,BLOCKS, and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofsimilarity using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

[0191] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. (See, e.g., Smith, T. etal. (1992) Protein Engineering 5:35-51.) The sequences disclosed in thisapplication have lengths of at least 49 nucleotides and have no morethan 12% uncalled bases (where N is recorded rather than A, C, G, or T).

[0192] The BLAST approach searched for matches between a query sequenceand a database sequence. BLAST evaluated the statistical significance ofany matches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁴ for peptides.

[0193] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam), and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for similarity.

[0194] Additionally, sequences identified from cDNA libraries may beanalyzed to identify those gene sequences encoding conserved proteinmotifs using an appropriate analysis program, e.g., BLOCKS. BLOCKS is aweighted matrix analysis algorithm based on short amino acid segments,or blocks, compiled from the PROSITE database. (Bairoch, A. et al.(1997) Nucleic Acids Res. 25:217-221.) The BLOCKS algorithm is usefulfor classifying genes with unknown functions. (Henikoff S. And HenikoffG. J., Nucleic Acids Research (1991) 19:6565-6572.) Blocks, which are3-60 amino acids in length, correspond to the most highly conservedregions of proteins. The BLOCKS algorithm compares a query sequence witha weighted scoring matrix of blocks in the BLOCKS database. Blocks inthe BLOCKS database are calibrated against protein sequences with knownfunctions from the SWISS-PROT database to determine the stochasticdistribution of matches. Similar databases such as PRINTS, a proteinfingerprint database, are also searchable using the BLOCKS algorithm.(Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37:417-424.)PRINTS is based on non-redundant sequences obtained from sources such asSWISS-PROT, GenBank, PIR, and NRL-3D.

[0195] The BLOCKS algorithm searches for matches between a querysequence and the BLOCKS or PRINTS database and evaluates the statisticalsignificance of any matches found. Matches from a BLOCKS or PRINTSsearch can be evaluated on two levels, local similarity and globalsimilarity. The degree of local similarity is measured by scores, andthe extent of global similarity is measured by score ranking andprobability values. A score of 1000 or greater for a BLOCKS match ofhighest ranking indicates that the match falls within the 0.5 percentilelevel of false positives when the matched block is calibrated againstSWISS-PROT. Likewise, a probability value of less than 1.0×10⁻³indicates that the match would occur by chance no more than one time inevery 1000 searches. Only those matches with a cutoff score of 1000 orgreater and a cutoff probability value of 1.0×10⁻³ or less areconsidered in the functional analyses of the protein sequences in theSequence Listing.

[0196] Nucleic and amino acid sequences of the Sequence Listing may alsobe analyzed using PFAM. PFAM is a Hidden Markov Model (HMM) basedprotocol useful in protein family searching. HMM is a probabilisticapproach which analyzes consensus primary structures of gene families.(See, e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.)

[0197] The PFAM database contains protein sequences of 527 proteinfamilies gathered from publicly available sources, e.g., SWISS-PROT andPROSITE. PFAM searches for well characterized protein domain familiesusing two high-quality alignment routines, seed alignment and fullalignment. (See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins28:405-420.) The seed alignment utilizes the hmmls program, a programthat searches for local matches, and a non-redundant set of the PFAMdatabase. The full alignment utilizes the hmmfs program, a program thatsearches for multiple fragments in long sequences, e.g., repeats andmotifs, and all sequences in the PFAM database. A result or score of 100“bits” can signify that it is 2¹⁰⁰-fold more likely that the sequence isa true match to the model or comparison sequence. Cutoff scores whichrange from 10 to 50 bits are generally used for individual proteinfamilies using the SWISS-PROT sequences as model or comparisonsequences.

[0198] Two other algorithms, SIGPEPT and TM, both based on the HMMalgorithm described above (see, e.g., Eddy, supra; and Sonnhammer,supra), identify potential signal sequences and transmembrane domains,respectively. SIGPEPT was created using protein sequences having signalsequence annotations derived from SWISS-PROT. It contains about 1413non-redundant signal sequences ranging in length from 14 to 36 aminoacid residues. TM was created similarly using transmembrane domainannotations. It contains about 453 non-redundant transmembrane sequencesencompassing 1579 transmembrane domain segments. Suitable HMM modelswere constructed using the above sequences and were refined with knownSWISS-PROT signal peptide sequences or transmembrane domain sequencesuntil a high correlation coefficient, a measurement of the correctnessof the analysis, was obtained. Using the protein sequences from theSWISS-PROT database as a test set, a cutoff score of 11 bits, asdetermined above, correlated with 91-94% true-positives and about 4.1%false-positives, yielding a correlation coefficient of about 0.87-0.90for SIGPEPT. A score of 11 bits for TM will typically give the followingresults: 75% true positives; 1.72% false positives; and a correlationcoefficient of 0.76. Each search evaluates the statistical significanceof any matches found and reports only those matches that score at least11 bits.

[0199] IV. Northern Analysis

[0200] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)

[0201] Analogous computer techniques applying BLAST are used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Genomics, Palo Alto Calif.). Thisanalysis is much faster than multiple membrane-based hybridizations. Inaddition, the sensitivity of the computer search can be modified todetermine whether any particular match is categorized as exact orsimilar.

[0202] The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0203] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

[0204] The results of Northern analysis are reported as a list oflibraries in which the transcript encoding GAPIP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0205] V. Extension of GAPIP Encoding Polynucleotides

[0206] The nucleic acid sequence of Incyte Clone 688183 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension of an antisense polynucleotide, and the other was synthesizedto initiate extension of a sense polynucleotide. Primers were used tofacilitate the extension of the known sequence “outward” generatingamplicons containing new unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 (National Biosciences), or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the target sequence at temperatures of about 68°C. to about 72° C. Any stretch of nucleotides which would result inhairpin structures and primer-primer dimerizations was avoided.

[0207] Selected human cDNA libraries (Life Technologies) were used toextend the sequence. If more than one extension is necessary or desired,additional sets of primers are designed to further extend the knownregion.

[0208] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Applied Biosystems) and thoroughlymixing the enzyme and reaction mix. PCR was performed using the DNAENGINE thermal cyclers (MJ Research), beginning with 40 pmol of eachprimer and the recommended concentrations of all other components of thekit, with the following parameters: Step 1 94° C. for 1 min (initialdenaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 min Step 4 94°C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 min Step 7Repeat steps 4 through 6 for an additional 15 cycles Step 8 94° C. for15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11Repeat steps 8 through 10 for an additional 12 cycles Step 12 72° C. for8 min Step 13 4° C. (and holding)

[0209] A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

[0210] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

[0211] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4for an additional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0212] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0213] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0214] VI. Labeling and Use of Individual Hybridization Probes

[0215] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (APB). An aliquot containing 10⁷ counts per minuteof the labeled probe is used in a typical membrane-based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases: AseI, BglII, EcoRI, PstI, Xba1, or PvuII (DuPont NEN,Boston, Mass.).

[0216] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots to film for severalhours, hybridization patterns are compared visually.

[0217] VII. Microarrays

[0218] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0219] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments suitablefor hybridization can be selected using software well known in the artsuch as LASERGENE. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

[0220] VIII. Complementary Polynucleotides

[0221] Sequences complementary to the GAPIP-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring GAPIP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of GAPIP. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the GAPIP-encoding transcript.

[0222] IX. Expression of GAPIP

[0223] Expression and purification of GAPIP is achieved using bacterialor virus-based expression systems. For expression of GAPIP in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express GAPIP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof GAPIP in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding GAPIP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

[0224] In most expression systems, GAPIP is synthesized as a fusionprotein with, e.g., glutathione S-transferase (GST) or a peptide epitopetag, such as FLAG or 6-His, permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Pharmacia, Piscataway, N.J.). Following purification, the GST moietycan be proteolytically cleaved from GAPIP at specifically engineeredsites. FLAG, an 8-amino acid peptide, enables immunoaffinitypurification using commercially available monoclonal and polyclonalanti-FLAG antibodies (Eastman Kodak, Rochester, N.Y.). 6-His, a stretchof six consecutive histidine residues, enables purification onmetal-chelate resins (QIAGEN Inc.). Methods for protein expression andpurification are discussed in Ausubel, F. M. et al. (1995 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., ch 10, 16. Purified GAPIP obtained by these methods canbe used directly in the following activity assay.

[0225] X. Demonstration of GAPIP Activity

[0226] Protease inhibitory activity of GAPIP is measured by theinhibition of hydrolysis by trypsin of appropriate synthetic peptidesubstrates conjugated with various chromogenic molecules in which thedegree of hydrolysis is quantitated by spectrophotometric (orfluorometric) absorption of the released chromophore (Beynon and Bondsupra, pp.25-55). Peptide substrates are selected for optimal activityusing prepared trypsin. Chromogens commonly used are 2-naphthylamine,4-nitroaniline, and furylacrylic acid. Assays are performed at ambienttemperature and contain an aliquot of trypsin, the appropriate substratein a suitable buffer, and serial dilutions of purified GAPIP. Reactionsare carried out in an optical cuvette and followed by theincrease/decrease in absorbance or fluorescence of the chromogenreleased during hydrolysis of the peptide substrate. The baselineabsorbance in the absence of GAPIP is proportional to the trypsinactivity in the assay. Reduction in absorbance is proportional to theGAPIP activity in the assay.

[0227] XI. Functional Assays

[0228] GAPIP function is assessed by expressing the sequences encodingGAPIP at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include pCMV SPORT (Life Technologies) and pCR 3.1 (Invitrogen,Carlsbad, Calif., both of which contain the cytomegalovirus promoter.5-10 μg of recombinant vector are transiently transfected into a humancell line, preferably of endothelial or hematopoietic origin, usingeither liposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP)(Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flowcytometry (FCM), an automated, laser optics-based technique, is used toidentify transfected cells expressing GFP or CD64-GFP, and to evaluateproperties, for example, their apoptotic state. FCM detects andquantifies the uptake of fluorescent molecules that diagnose eventspreceding or coincident with cell death. These events include changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry,Oxford, New York, N.Y.

[0229] The influence of GAPIP on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodingGAPIP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed onthe surface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding GAPIP and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

[0230] XII. Production of GAPIP Specific Antibodies

[0231] GAPIP substantially purified using polyacrylamide gelelectrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182:488495), or other purification techniques, is used toimmunize rabbits and to produce antibodies using standard protocols.

[0232] Alternatively, the GAPIP amino acid sequence is analyzed usingLASERGENE software to 25 determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubelsupra, ch. 11.)

[0233] Typically, oligopeptides 15 residues in length are synthesizedusing an Applied Biosystems 30 peptide synthesizer Model 431A usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

[0234] XIII. Purification of Naturally Occurring GAPIP Using SpecificAntibodies

[0235] Naturally occurring or recombinant GAPIP is substantiallypurified by immunoaffinity chromatography using antibodies specific forGAPIP. An immunoaffinity column is constructed by covalently couplinganti-GAPIP antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (APB). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0236] Media containing GAPIP are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of GAPIP (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/GAPIP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGAPIP is collected.

[0237] XIV. Identification of Molecules Which Interact with GAPIP

[0238] GAPIP, or biologically active fragments thereof, are labeled with125I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled GAPIP, washed, and anywells with labeled GAPIP complex are assayed. Data obtained usingdifferent concentrations of GAPIP are used to calculate values for thenumber, affinity, and association of GAPIP with the candidate molecules.

[0239] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 5 942 amino acids amino acid single linear UTRSNOT02 688183 1 Met LeuLeu Leu Leu Gly Leu Cys Leu Gly Leu Ser Leu Cys Val 5 10 15 Gly Ser GlnGlu Glu Ala Gln Ser Trp Gly His Ser Ser Glu Gln 20 25 30 Asp Gly Leu ArgVal Pro Arg Gln Val Arg Leu Leu Gln Arg Leu 35 40 45 Lys Thr Lys Pro LeuMet Thr Glu Phe Ser Val Lys Ser Thr Ile 50 55 60 Ile Ser Arg Tyr Ala PheThr Thr Val Ser Cys Arg Met Leu Asn 65 70 75 Arg Ala Ser Glu Asp Gln AspIle Glu Phe Gln Met Gln Ile Pro 80 85 90 Ala Ala Ala Phe Ile Thr Asn PheThr Met Leu Ile Gly Asp Lys 95 100 105 Val Tyr Gln Gly Glu Ile Thr GluArg Glu Lys Lys Ser Gly Asp 110 115 120 Arg Val Lys Glu Lys Arg Asn LysThr Thr Glu Glu Asn Gly Glu 125 130 135 Lys Gly Thr Glu Ile Phe Arg AlaSer Ala Val Ile Pro Ser Lys 140 145 150 Asp Lys Ala Ala Phe Phe Leu SerTyr Glu Glu Leu Leu Gln Arg 155 160 165 Arg Leu Gly Lys Tyr Glu His SerIle Ser Val Arg Pro Gln Gln 170 175 180 Leu Ser Gly Arg Leu Ser Val AspVal Asn Ile Leu Glu Ser Ala 185 190 195 Gly Ile Ala Ser Leu Glu Val LeuPro Leu His Asn Ser Arg Gln 200 205 210 Arg Gly Ser Gly Arg Gly Glu AspAsp Ser Gly Pro Pro Pro Ser 215 220 225 Thr Val Ile Asn Gln Asn Glu ThrPhe Ala Asn Ile Ile Phe Lys 230 235 240 Pro Thr Val Val Gln Gln Ala ArgIle Ala Gln Asn Gly Ile Leu 245 250 255 Gly Asp Phe Ile Ile Arg Tyr AspVal Asn Arg Glu Gln Ser Ile 260 265 270 Gly Asp Ile Gln Val Leu Asn GlyTyr Phe Val His Tyr Phe Ala 275 280 285 Pro Lys Asp Leu Pro Pro Leu ProLys Asn Val Val Phe Val Leu 290 295 300 Asp Ser Ser Ala Ser Met Val GlyThr Lys Leu Arg Gln Thr Lys 305 310 315 Asp Ala Leu Phe Thr Ile Leu HisAsp Leu Arg Pro Gln Asp Arg 320 325 330 Phe Ser Ile Ile Gly Phe Ser AsnArg Ile Lys Val Trp Lys Asp 335 340 345 His Leu Ile Ser Val Thr Pro AspSer Ile Arg Asp Gly Lys Val 350 355 360 Tyr Ile His His Met Ser Pro ThrGly Gly Thr Asp Ile Asn Gly 365 370 375 Ala Leu Gln Arg Ala Ile Arg LeuLeu Asn Lys Tyr Val Ala His 380 385 390 Ser Gly Ile Gly Asp Arg Ser ValSer Leu Ile Val Phe Leu Thr 395 400 405 Asp Gly Lys Pro Thr Val Gly GluThr His Thr Leu Lys Ile Leu 410 415 420 Asn Asn Thr Arg Glu Ala Ala ArgGly Gln Val Cys Ile Phe Thr 425 430 435 Ile Gly Ile Gly Asn Asp Val AspPhe Arg Leu Leu Glu Lys Leu 440 445 450 Ser Leu Glu Asn Cys Gly Leu ThrArg Arg Val His Glu Glu Glu 455 460 465 Asp Ala Gly Ser Gln Leu Ile GlyPhe Tyr Asp Glu Ile Arg Thr 470 475 480 Pro Leu Leu Ser Asp Ile Arg IleAsp Tyr Pro Pro Ser Ser Val 485 490 495 Val Gln Ala Thr Lys Thr Leu PhePro Asn Tyr Phe Asn Gly Ser 500 505 510 Glu Ile Ile Ile Ala Gly Lys LeuVal Asp Arg Lys Leu Asp His 515 520 525 Leu His Val Glu Val Thr Ala SerAsn Ser Lys Lys Phe Ile Ile 530 535 540 Leu Lys Thr Asp Val Pro Val ArgPro Gln Lys Ala Gly Lys Asp 545 550 555 Val Thr Gly Ser Pro Arg Pro GlyGly Asp Gly Glu Gly Asp Thr 560 565 570 Asn His Ile Glu Arg Leu Trp SerTyr Leu Thr Thr Lys Glu Leu 575 580 585 Leu Ser Ser Trp Leu Gln Ser AspAsp Glu Pro Glu Lys Glu Arg 590 595 600 Leu Arg Gln Arg Ala Gln Ala LeuAla Val Ser Tyr Arg Phe Leu 605 610 615 Thr Pro Phe Thr Ser Met Lys LeuArg Gly Pro Val Pro Arg Met 620 625 630 Asp Gly Leu Glu Glu Ala His GlyMet Ser Ala Ala Met Gly Pro 635 640 645 Glu Pro Val Val Gln Ser Val ArgGly Ala Gly Thr Gln Pro Gly 650 655 660 Pro Leu Leu Lys Lys Pro Tyr GlnPro Arg Ile Lys Ile Ser Lys 665 670 675 Thr Ser Val Asp Gly Asp Pro HisPhe Val Val Asp Phe Pro Leu 680 685 690 Ser Arg Leu Thr Val Cys Phe AsnIle Asp Gly Gln Pro Gly Asp 695 700 705 Ile Leu Arg Leu Val Ser Asp HisArg Asp Ser Gly Val Thr Val 710 715 720 Asn Gly Glu Leu Ile Gly Ala ProAla Pro Pro Asn Gly His Lys 725 730 735 Lys Gln Arg Thr Tyr Leu Arg ThrIle Thr Ile Leu Ile Asn Lys 740 745 750 Pro Glu Arg Ser Tyr Leu Glu IleThr Pro Ser Arg Val Ile Leu 755 760 765 Asp Gly Gly Asp Arg Leu Val LeuPro Cys Asn Gln Ser Val Val 770 775 780 Val Gly Ser Trp Gly Leu Glu ValSer Val Ser Ala Asn Ala Asn 785 790 795 Val Thr Val Thr Ile Gln Gly SerIle Ala Phe Val Ile Leu Ile 800 805 810 His Leu Tyr Lys Lys Pro Ala ProPhe Gln Arg His His Leu Gly 815 820 825 Phe Tyr Ile Ala Asn Ser Glu GlyLeu Ser Ser Asn Cys His Gly 830 835 840 Leu Leu Gly Gln Phe Leu Asn GlnAsp Ala Arg Leu Thr Glu Asp 845 850 855 Pro Ala Gly Pro Ser Gln Asn LeuThr His Pro Leu Leu Leu Gln 860 865 870 Val Gly Glu Gly Pro Glu Ala ValLeu Thr Val Lys Gly His Gln 875 880 885 Val Pro Val Val Trp Lys Gln ArgLys Ile Tyr Asn Gly Glu Glu 890 895 900 Gln Ile Asp Cys Trp Phe Ala ArgAsn Asn Ala Ala Lys Leu Ile 905 910 915 Asp Gly Glu Tyr Lys Asp Tyr LeuAla Ser His Pro Phe Asp Thr 920 925 930 Gly Met Thr Leu Gly Arg Gly MetSer Arg Glu Leu 935 940 3636 base pairs nucleic acid single linearUTRSNOT02 688183 2 CCCTGAGAGC GTCCCGCAGT GGCTGGAGCC CTGGGCGCTGCAAACGTGTC CCGCCGGGTC 60 CCCGAGCGTC CCGCGCCCTC GCCCCGCCAT GCTCCTGCTGCTGGGGCTGT GCCTGGGGCT 120 GTCCCTGTGT GTGGGGTCGC AGGAAGAGGC GCAGAGCTGGGGCCACTCTT CGGAGCAGGA 180 TGGACTCAGG GTCCCGAGGC AAGTCAGACT GTTGCAGAGGCTGAAAACCA AACCTTTGAT 240 GACAGAATTC TCAGTGAAGT CTACCATCAT TTCCCGTTATGCCTTCACTA CGGTTTCCTG 300 CAGAATGCTG AACAGAGCTT CTGAAGACCA GGACATTGAGTTCCAGATGC AGATTCCAGC 360 TGCAGCTTTC ATCACCAACT TCACTATGCT TATTGGAGACAAGGTGTATC AGGGCGAAAT 420 TACAGAGAGA GAAAAGAAGA GTGGTGATAG GGTAAAAGAGAAAAGGAATA AAACCACAGA 480 AGAAAATGGA GAGAAGGGGA CTGAAATATT CAGAGCTTCTGCAGTGATTC CCAGCAAGGA 540 CAAAGCCGCC TTTTTCCTGA GTTATGAGGA GCTTCTGCAGAGGCGCCTGG GCAAGTACGA 600 GCACAGCATC AGCGTGCGGC CCCAGCAGCT GTCCGGGAGGCTGAGCGTGG ACGTGAATAT 660 CCTGGAGAGC GCGGGCATCG CATCCCTGGA GGTGCTGCCGCTTCACAACA GCAGGCAGAG 720 GGGCAGTGGG CGCGGGGAAG ATGATTCTGG GCCTCCCCCATCTACTGTCA TTAACCAAAA 780 TGAAACATTT GCCAACATAA TTTTTAAACC TACTGTAGTACAACAAGCCA GGATTGCCCA 840 GAATGGAATT TTGGGAGACT TTATCATTAG ATATGACGTCAATAGAGAAC AGAGCATTGG 900 GGACATCCAG GTTCTAAATG GCTATTTTGT GCACTACTTTGCTCCTAAAG ACCTTCCTCC 960 TTTACCCAAG AATGTGGTAT TCGTGCTTGA CAGCAGTGCTTCTATGGTGG GAACCAAACT 1020 CCGGCAGACC AAGGATGCCC TCTTCACAAT TCTCCATGACCTCCGACCCC AGGACCGTTT 1080 CAGTATCATT GGATTTTCCA ACCGGATCAA AGTATGGAAGGACCACTTGA TATCAGTCAC 1140 TCCAGACAGC ATCAGGGATG GGAAAGTGTA CATTCACCATATGTCACCCA CTGGAGGCAC 1200 AGACATCAAC GGGGCCCTGC AGAGGGCCAT CAGGCTCCTCAACAAGTACG TGGCCCACAG 1260 TGGCATTGGA GACCGGAGCG TGTCCCTCAT CGTCTTCCTGACGGATGGGA AGCCCACGGT 1320 CGGGGAGACG CACACCCTCA AGATCCTCAA CAACACCCGAGAGGCCGCCC GAGGCCAAGT 1380 CTGCATCTTC ACCATTGGCA TCGGCAACGA CGTGGACTTCAGGCTGCTGG AGAAACTGTC 1440 GCTGGAGAAC TGTGGCCTCA CACGGCGCGT GCACGAGGAGGAGGACGCAG GCTCGCAGCT 1500 CATCGGGTTC TACGATGAAA TCAGGACCCC GCTCCTCTCTGACATCCGCA TCGATTATCC 1560 CCCCAGCTCA GTGGTGCAGG CCACCAAGAC CCTGTTCCCCAACTACTTCA ACGGCTCGGA 1620 GATCATCATT GCGGGGAAGC TGGTGGACAG GAAGCTGGATCACCTGCACG TGGAGGTCAC 1680 CGCCAGCAAC AGTAAGAAAT TCATCATCCT GAAGACAGATGTGCCTGTGC GGCCTCAGAA 1740 GGCAGGGAAA GATGTCACAG GAAGCCCCAG GCCTGGAGGCGATGGAGAGG GGGACACCAA 1800 CCACATCGAG CGTCTCTGGA GCTACCTCAC CACAAAGGAGCTGCTGAGCT CCTGGCTGCA 1860 AAGTGACGAT GAACCGGAGA AGGAGCGGCT GCGGCAGCGGGCCCAGGCCC TGGCTGTGAG 1920 CTACCGCTTC CTCACTCCCT TCACCTCCAT GAAGCTGAGGGGGCCGGTCC CACGCATGGA 1980 TGGCCTGGAG GAGGCCCACG GCATGTCGGC TGCCATGGGACCCGAACCGG TGGTGCAGAG 2040 CGTGCGAGGA GCTGGCACGC AGCCAGGGCC TTTGCTCAAGAAGCCATACC AGCCAAGAAT 2100 TAAAATCTCT AAAACATCAG TGGATGGTGA TCCCCACTTTGTTGTGGATT TCCCCCTGAG 2160 CAGACTCACC GTGTGCTTCA ACATTGATGG GCAGCCCGGGGACATCCTCA GGCTGGTCTC 2220 TGATCACAGG GACTCTGGTG TCACAGTGAA CGGAGAGTTAATTGGGGCAC CCGCCCCTCC 2280 AAATGGCCAC AAGAAACAGC GCACTTACTT GCGCACTATCACCATCCTCA TCAACAAGCC 2340 AGAGAGATCT TATCTCGAGA TCACACCGAG CAGAGTCATCTTGGATGGTG GGGACAGACT 2400 GGTGCTCCCC TGCAACCAGA GTGTGGTGGT GGGGAGCTGGGGGCTGGAGG TGTCCGTGTC 2460 TGCCAACGCC AATGTCACCG TCACCATCCA GGGCTCCATAGCCTTTGTCA TCCTCATCCA 2520 CCTCTACAAA AAGCCGGCGC CCTTCCAGCG ACACCACCTGGGTTTCTACA TTGCCAACAG 2580 CGAGGGCCTT TCCAGCAACT GCCACGGACT GCTGGGTCAGTTCCTGAATC AGGATGCCAG 2640 ACTCACAGAA GACCCTGCAG GGCCCAGCCA GAACCTCACTCACCCTCTGC TCCTTCAGGT 2700 GGGAGAGGGG CCTGAGGCCG TCCTAACAGT GAAAGGCCACCAAGTCCCAG TGGTCTGGAA 2760 GCAAAGGAAG ATTTACAACG GGGAAGAGCA GATAGACTGCTGGTTTGCCA GGAACAATGC 2820 CGCCAAACTG ATTGACGGGG AGTACAAGGA TTACCTGGCATCCCATCCAT TTGACACAGG 2880 GATGACACTT GGCCGGGGAA TGTCCAGGGA GCTCTGAAGCTGGCAGCCTT AAAGATGCAA 2940 GTGCATGAAG GACAGTGATG TGGGGAGGCC GTGGGGCAGCTCTTTTCATG GCTTGTACAC 3000 GCCTCAGCTC CTGGCAATTA GCTGGACTCC ATGACCCACCCCTGGTGCAG CATAGATCCG 3060 ACGTCTGTCT GGGCGAAGGG TAGGGGTGGG TAGGGGCGGGAAGCCTGAGT GCAAATGTCA 3120 TTTCCCTCTA CTGCCTCTTC CTGCCTCTCC CCACCCTGCCCACATCCACA GAGGGGAGAG 3180 AAGGGTCATA GCTAAATGCA ACAAAGTCTG TATCTTGTCCCAACCTGCTT TTCTGTTCTG 3240 TTAGCATATC ATAAAGTAAG CCTTTCTGGT GAAGGAAGGTTGCTATGAAA CTTTTTTTCT 3300 TGGTGGAAAT GGCCAAGTTT AGGCACTCTG CTTTTTGCCTTACACTAATG CTTAGAAAGC 3360 TGTCTTTTCA GTGGTGTTGC AGCCCCCAGA TGTGTGGCCAACCTCTGCTG CAAAGGAATC 3420 TCTTGCTGAG TCCAGGCCAC CAATCAGGCA AATAGCCCATACATTTGATC GTTGTAAACC 3480 ATGAAGTCTT TTCTTGCAAG ACGTTTTTCT TCTGCTGTGGTATCTTGCCC TTAAAAATTA 3540 GTTTTCATTA AAAAGAAATT TGATTGAAAA TTAAAAAAAAATAAAAAAAA AAGAAAAAAA 3600 AAAAGAAAGA AAAAATAAAA AAAAAAAAAA AAAAAA 3636946 amino acids amino acid single linear GENEBANK gi33985 3 Met Lys ArgLeu Thr Cys Phe Phe Ile Cys Phe Phe Leu Ser Glu 5 10 15 Val Ser Gly PheGlu Ile Pro Ile Asn Gly Leu Ser Glu Phe Val 20 25 30 Asp Tyr Glu Asp LeuVal Glu Leu Ala Pro Gly Lys Phe Gln Leu 35 40 45 Val Ala Glu Asn Arg ArgTyr Gln Arg Ser Leu Pro Gly Glu Ser 50 55 60 Glu Glu Met Met Glu Glu ValAsp Gln Val Thr Leu Tyr Ser Tyr 65 70 75 Lys Val Gln Ser Thr Ile Thr SerArg Met Ala Thr Thr Met Ile 80 85 90 Gln Ser Lys Val Val Asn Asn Ser ProGln Pro Gln Asn Val Val 95 100 105 Phe Asp Val Gln Ile Pro Lys Gly AlaPhe Ile Ser Asn Phe Ser 110 115 120 Met Thr Val Asp Gly Lys Thr Phe ArgSer Ser Ile Lys Glu Lys 125 130 135 Thr Val Gly Arg Ala Leu Tyr Ala GlnAla Arg Ala Lys Gly Lys 140 145 150 Thr Ala Gly Leu Val Arg Ser Ser AlaLeu Asp Met Glu Asn Phe 155 160 165 Arg Thr Glu Val Asn Val Leu Pro GlyAla Lys Val Gln Phe Glu 170 175 180 Leu His Tyr Gln Glu Val Lys Trp ArgLys Leu Gly Ser Tyr Glu 185 190 195 His Arg Ile Tyr Leu Gln Pro Gly ArgLeu Ala Lys His Leu Glu 200 205 210 Val Asp Val Trp Val Ile Glu Pro GlnGly Leu Arg Phe Leu His 215 220 225 Val Pro Asp Thr Phe Glu Gly His PheAsp Gly Val Pro Val Ile 230 235 240 Ser Lys Gly Gln Gln Lys Ala His ValSer Phe Lys Pro Thr Val 245 250 255 Ala Gln Gln Arg Ile Cys Pro Ser CysArg Glu Thr Ala Val Asp 260 265 270 Gly Glu Leu Val Val Leu Tyr Asp ValLys Arg Glu Glu Lys Ala 275 280 285 Gly Glu Leu Glu Val Phe Asn Gly TyrPhe Val His Phe Phe Ala 290 295 300 Pro Asp Asn Leu Asp Pro Ile Pro LysAsn Ile Leu Phe Val Ile 305 310 315 Asp Val Ser Gly Ser Met Trp Gly ValLys Met Lys Gln Thr Val 320 325 330 Glu Ala Met Lys Thr Ile Leu Asp AspLeu Arg Ala Glu Asp His 335 340 345 Phe Ser Val Ile Asp Phe Asn Gln AsnIle Arg Thr Trp Arg Asn 350 355 360 Asp Leu Phe Gln Leu Gln Lys His ArgLeu Gln Ile Ala Lys Arg 365 370 375 Tyr Ile Glu Lys Ile Gln Pro Ser GlyGly Thr Asn Ile Asn Glu 380 385 390 Ala Leu Leu Arg Ala Ile Phe Ile LeuAsn Glu Ala Asn Asn Leu 395 400 405 Gly Leu Leu Asp Pro Asn Ser Val SerLeu Ile Ile Leu Val Ser 410 415 420 Asp Gly Asp Pro Thr Val Gly Glu LeuLys Leu Ser Lys Ile Gln 425 430 435 Lys Asn Val Lys Glu Asn Ile Gln AspAsn Ile Ser Leu Phe Ser 440 445 450 Leu Gly Met Gly Phe Asp Val Asp TyrAsp Phe Leu Lys Arg Leu 455 460 465 Ser Asn Glu Asn His Gly Ile Ala GlnArg Ile Tyr Gly Asn Gln 470 475 480 Asp Thr Ser Ser Gln Leu Lys Lys PheTyr Asn Gln Val Ser Thr 485 490 495 Pro Leu Leu Arg Asn Val Gln Phe AsnTyr Pro His Thr Ser Val 500 505 510 Thr Asp Val Thr Gln Asn Asn Phe HisAsn Tyr Phe Gly Gly Ser 515 520 525 Glu Ile Val Val Ala Gly Lys Phe AspPro Ala Lys Leu Asp Gln 530 535 540 Ile Glu Ser Val Ile Thr Ala Thr SerAla Asn Thr Gln Leu Val 545 550 555 Leu Glu Thr Leu Ala Gln Met Asp AspLeu Gln Asp Phe Leu Ser 560 565 570 Lys Asp Lys His Ala Asp Pro Asp PheThr Arg Lys Leu Trp Ala 575 580 585 Tyr Leu Thr Ile Asn Gln Leu Leu AlaGlu Arg Ser Leu Ala Pro 590 595 600 Thr Ala Ala Ala Lys Arg Arg Ile ThrArg Ser Ile Leu Gln Met 605 610 615 Ser Leu Asp His His Ile Val Thr ProLeu Thr Ser Leu Val Ile 620 625 630 Glu Asn Glu Ala Gly Asp Glu Arg MetLeu Ala Asp Ala Pro Pro 635 640 645 Gln Asp Pro Ser Cys Cys Ser Gly AlaLeu Tyr Tyr Gly Ser Lys 650 655 660 Val Val Pro Asp Ser Thr Pro Ser TrpAla Asn Pro Ser Pro Thr 665 670 675 Pro Val Ile Ser Met Leu Ala Gln GlySer Gln Val Leu Glu Ser 680 685 690 Thr Pro Pro Pro His Val Met Arg ValGlu Asn Asp Pro His Phe 695 700 705 Ile Ile Tyr Leu Pro Lys Ser Gln LysAsn Ile Cys Phe Asn Ile 710 715 720 Asp Ser Glu Pro Gly Lys Ile Leu AsnLeu Val Ser Asp Pro Glu 725 730 735 Ser Gly Ile Val Val Asn Gly Gln LeuVal Gly Ala Lys Lys Pro 740 745 750 Asn Asn Gly Lys Leu Ser Thr Tyr PheGly Lys Leu Gly Phe Tyr 755 760 765 Phe Gln Ser Glu Asp Ile Lys Ile GluIle Ser Thr Glu Thr Ile 770 775 780 Thr Leu Ser His Gly Ser Ser Thr PheSer Leu Ser Trp Ser Asp 785 790 795 Thr Ala Gln Val Thr Asn Gln Arg ValGln Ile Ser Val Lys Lys 800 805 810 Glu Lys Val Val Thr Ile Thr Leu AspLys Glu Met Ser Phe Ser 815 820 825 Val Leu Leu His Arg Val Trp Lys LysHis Pro Val Asn Val Asp 830 835 840 Phe Leu Gly Ile Tyr Ile Pro Pro ThrAsn Lys Phe Ser Pro Lys 845 850 855 Ala His Gly Leu Ile Gly Gln Phe MetGln Glu Pro Lys Ile His 860 865 870 Ile Phe Asn Glu Arg Pro Gly Lys AspPro Glu Lys Pro Glu Ala 875 880 885 Ser Met Glu Val Lys Gly Gln Lys LeuIle Ile Thr Arg Gly Leu 890 895 900 Gln Lys Asp Tyr Arg Thr Asp Leu ValPhe Gly Thr Asp Val Thr 905 910 915 Cys Trp Phe Val His Asn Ser Gly LysGly Phe Ile Asp Gly His 920 925 930 Tyr Lys Asp Tyr Phe Val Pro Gln LeuTyr Ser Phe Leu Lys Arg 935 940 945 Pro 911 amino acids amino acidsingle linear GENEBABK gi33989 4 Met Asp Gly Ala Met Gly Pro Arg Gly LeuLeu Leu Cys Met Tyr 5 10 15 Leu Val Ser Leu Leu Ile Leu Gln Ala Met ProAla Leu Gly Ser 20 25 30 Ala Thr Gly Arg Ser Lys Ser Ser Glu Lys Arg GlnAla Val Asp 35 40 45 Thr Ala Val Asp Gly Val Phe Ile Arg Ser Leu Lys ValAsn Cys 50 55 60 Lys Val Thr Ser Arg Phe Ala His Tyr Val Val Thr Ser GlnVal 65 70 75 Val Asn Thr Ala Asn Glu Ala Arg Glu Val Ala Phe Asp Leu Glu80 85 90 Ile Pro Lys Thr Ala Phe Ile Ser Asp Phe Ala Val Thr Ala Asp 95100 105 Gly Asn Ala Phe Ile Gly Asp Ile Lys Asp Lys Val Thr Ala Trp 110115 120 Lys Gln Tyr Arg Lys Ala Ala Ile Ser Gly Glu Asn Ala Gly Leu 125130 135 Val Arg Ala Ser Gly Arg Thr Met Glu Gln Phe Thr Ile His Leu 140145 150 Thr Val Asn Pro Gln Ser Lys Val Thr Phe Gln Leu Thr Tyr Glu 155160 165 Glu Val Leu Lys Arg Asn His Met Gln Tyr Glu Ile Val Ile Lys 170175 180 Val Lys Pro Lys Gln Leu Val His His Phe Glu Ile Asp Val Asp 185190 195 Ile Phe Glu Pro Gln Gly Ile Ser Lys Leu Asp Ala Gln Ala Ser 200205 210 Phe Leu Pro Lys Glu Leu Ala Ala Gln Thr Ile Lys Lys Ser Phe 215220 225 Ser Gly Lys Lys Gly His Val Leu Phe Arg Pro Thr Val Ser Gln 230235 240 Gln Gln Ser Cys Pro Thr Cys Ser Thr Ser Leu Leu Asn Gly His 245250 255 Phe Lys Val Thr Tyr Asp Val Thr Arg Asp Glu Ile Cys Asp Leu 260265 270 Leu Val Ala Asn Asn His Phe Ala His Phe Phe Ala Pro Gln Asn 275280 285 Leu Thr Asn Met Asn Lys Asn Val Val Phe Val Ile Asp Ile Ser 290295 300 Gly Ser Met Arg Gly Gln Lys Val Lys Gln Thr Lys Glu Ala Leu 305310 315 Leu Lys Ile Leu Gly Asp Met Gln Pro Gly Asp Tyr Phe Asp Leu 320325 330 Val Leu Phe Gly Thr Arg Val Gln Ser Trp Lys Gly Ser Leu Val 335340 345 Gln Ala Ser Glu Ala Asn Leu Gln Ala Ala Gln Asp Phe Val Arg 350355 360 Gly Phe Ser Leu Asp Glu Ala Thr Asn Leu Asn Gly Gly Leu Leu 365370 375 Arg Gly Ile Glu Ile Leu Asn Gln Val Gln Glu Ser Leu Pro Glu 380385 390 Leu Ser Asn His Ala Ser Ile Leu Ile Met Leu Thr Asp Gly Asp 395400 405 Pro Thr Glu Gly Val Thr Asp Arg Ser Gln Ile Leu Lys Asn Val 410415 420 Arg Asn Ala Ile Arg Gly Arg Phe Pro Leu Tyr Asn Leu Gly Phe 425430 435 Gly His Asn Val Asp Phe Asn Phe Leu Glu Val Met Ser Met Glu 440445 450 Asn Asn Gly Arg Ala Gln Arg Ile Tyr Glu Asp His Asp Ala Thr 455460 465 Gln Gln Leu Gln Gly Phe Tyr Ser Gln Val Ala Lys Pro Leu Leu 470475 480 Val Asp Val Asp Leu Gln Tyr Pro Gln Asp Ala Val Leu Ala Leu 485490 495 Thr Gln Asn His His Lys Gln Tyr Tyr Glu Gly Ser Glu Ile Val 500505 510 Val Ala Gly Arg Ile Ala Asp Asn Lys Gln Ser Ser Phe Lys Ala 515520 525 Asp Val Gln Ala His Gly Glu Gly Gln Glu Phe Ser Ile Thr Cys 530535 540 Leu Val Asp Glu Glu Glu Met Lys Lys Leu Leu Arg Glu Arg Gly 545550 555 His Met Leu Glu Asn His Val Glu Arg Leu Trp Ala Tyr Leu Thr 560565 570 Ile Gln Glu Leu Leu Ala Lys Arg Met Lys Val Asp Arg Glu Val 575580 585 Arg Ala Asn Leu Ser Ser Gln Ala Leu Arg Met Ser Leu Asp Tyr 590595 600 Gly Phe Val Thr Pro Leu Thr Ser Met Ser Ile Arg Gly Met Ala 605610 615 Asp Gln Asp Gly Leu Lys Pro Thr Ile Asp Lys Pro Ser Glu Asp 620625 630 Ser Pro Pro Leu Glu Met Leu Gly Pro Arg Arg Thr Phe Val Leu 635640 645 Ser Ala Leu Gln Pro Ser Pro Thr His Ser Ser Ser Asn Thr Gln 650655 660 Arg Leu Pro Asp Arg Val Thr Gly Val Asp Thr Asp Pro His Phe 665670 675 Ile Ile His Val Pro Gln Lys Glu Asp Thr Leu Cys Phe Asn Ile 680685 690 Asn Glu Glu Pro Gly Val Ile Leu Ser Leu Val Gln Asp Pro Asn 695700 705 Thr Gly Phe Ser Val Asn Gly Gln Leu Ile Gly Asn Lys Ala Arg 710715 720 Ser Pro Gly Gln His Asp Gly Thr Tyr Phe Gly Arg Leu Gly Ile 725730 735 Ala Asn Pro Ala Thr Asp Phe Gln Leu Glu Val Thr Pro Gln Asn 740745 750 Ile Thr Leu Asn Pro Gly Phe Gly Gly Pro Val Phe Ser Trp Arg 755760 765 Asp Gln Ala Val Leu Arg Gln Asp Gly Val Val Val Thr Ile Asn 770775 780 Lys Lys Arg Asn Leu Val Val Ser Val Asp Asp Gly Gly Thr Phe 785790 795 Glu Val Val Leu His Arg Val Trp Lys Gly Ser Ser Val His Gln 800805 810 Asp Phe Leu Gly Phe Tyr Val Leu Asp Ser His Arg Met Ser Ala 815820 825 Arg Thr His Gly Leu Leu Gly Gln Phe Phe His Pro Ile Gly Phe 830835 840 Glu Val Ser Asp Ile His Pro Gly Ser Asp Pro Thr Lys Pro Asp 845850 855 Ala Thr Met Val Val Arg Asn Arg Arg Leu Thr Val Thr Arg Gly 860865 870 Leu Gln Lys Asp Tyr Ser Lys Asp Pro Trp His Gly Ala Glu Val 875880 885 Ser Cys Trp Phe Ile His Asn Asn Gly Ala Gly Leu Ile Asp Gly 890895 900 Ala Tyr Thr Asp Tyr Ile Val Pro Asp Ile Phe 905 910 885 aminoacids amino acid single linear GENEBANK gi288563 5 Met Val Ala Leu SerHis Leu Gly Ser Ala Leu Gln Leu Gly Ser 5 10 15 Leu Cys Phe Pro Arg SerPro Phe Arg Leu Leu Gly Lys Arg Ser 20 25 30 Leu Pro Glu Gly Val Ala AsnGly Ile Glu Val Tyr Ser Thr Lys 35 40 45 Ile Asn Ser Lys Val Thr Ser ArgPhe Ala His Asn Val Val Thr 50 55 60 Met Arg Ala Val Asn Arg Ala Asp ThrAla Lys Glu Val Ser Phe 65 70 75 Asp Val Glu Leu Pro Lys Thr Ala Phe IleThr Asn Phe Thr Leu 80 85 90 Thr Ile Asp Gly Val Thr Tyr Pro Gly Asn ValLys Glu Lys Glu 95 100 105 Val Ala Lys Lys Gln Tyr Glu Lys Ala Val SerGln Gly Lys Thr 110 115 120 Ala Gly Leu Val Lys Ala Ser Gly Arg Lys LeuGlu Lys Phe Thr 125 130 135 Val Ser Val Asn Val Ala Ala Gly Ser Lys ValThr Phe Glu Leu 140 145 150 Thr Tyr Glu Glu Leu Leu Lys Arg His Lys GlyLys Tyr Glu Met 155 160 165 Tyr Leu Lys Val Gln Pro Lys Gln Leu Val LysHis Phe Glu Ile 170 175 180 Glu Val Asp Ile Phe Glu Pro Gln Gly Ile SerMet Leu Asp Ala 185 190 195 Glu Ala Ser Phe Ile Thr Asn Asp Leu Leu GlySer Ala Leu Thr 200 205 210 Lys Ser Phe Ser Gly Lys Lys Gly His Val SerPhe Lys Pro Ser 215 220 225 Leu Asp Gln Gln Arg Ser Cys Pro Thr Cys ThrAsp Ser Leu Leu 230 235 240 Asn Gly Asp Phe Thr Ile Thr Tyr Asp Val AsnArg Glu Ser Pro 245 250 255 Gly Asn Val Gln Ile Val Asn Gly Tyr Phe ValHis Phe Phe Ala 260 265 270 Pro Gln Gly Leu Pro Val Val Pro Lys Asn ValAla Phe Val Ile 275 280 285 Asp Ile Ser Gly Ser Met Ala Gly Arg Lys LeuGlu Gln Thr Lys 290 295 300 Glu Ala Leu Leu Arg Ile Leu Glu Asp Met LysGlu Glu Asp Tyr 305 310 315 Leu Asn Phe Ile Leu Phe Ser Gly Asp Val SerThr Trp Lys Glu 320 325 330 His Leu Val Gln Ala Thr Pro Glu Asn Leu GlnGlu Ala Arg Thr 335 340 345 Phe Val Lys Ser Met Glu Asp Lys Gly Met ThrAsn Ile Asn Asp 350 355 360 Gly Leu Leu Arg Gly Ile Ser Met Leu Asn LysAla Arg Glu Glu 365 370 375 His Arg Ile Pro Glu Arg Ser Thr Ser Ile ValIle Met Leu Thr 380 385 390 Asp Gly Asp Ala Asn Val Gly Glu Ser Arg ProGlu Lys Ile Gln 395 400 405 Glu Asn Val Arg Asn Ala Ile Gly Gly Lys PhePro Leu Tyr Asn 410 415 420 Leu Gly Phe Gly Asn Asn Leu Asn Tyr Asn PheLeu Glu Asn Met 425 430 435 Ala Leu Glu Asn His Gly Phe Ala Arg Arg IleTyr Glu Asp Ser 440 445 450 Asp Ala Asp Leu Gln Leu Gln Gly Phe Tyr GluGlu Val Ala Asn 455 460 465 Pro Leu Leu Thr Gly Val Glu Met Glu Tyr ProGlu Asn Ala Ile 470 475 480 Leu Asp Leu Thr Gln Asn Thr Tyr Gln His PheTyr Asp Gly Ser 485 490 495 Glu Ile Val Val Ala Gly Arg Leu Val Asp GluAsp Met Asn Ser 500 505 510 Phe Lys Ala Asp Val Lys Gly His Gly Ala ThrAsn Asp Leu Thr 515 520 525 Phe Thr Glu Glu Val Asp Met Lys Glu Met GluLys Ala Leu Gln 530 535 540 Glu Arg Asp Tyr Ile Phe Gly Asn Tyr Ile GluArg Leu Trp Ala 545 550 555 Tyr Leu Thr Ile Glu Gln Leu Leu Glu Lys ArgLys Asn Ala His 560 565 570 Gly Glu Glu Lys Glu Asn Leu Thr Ala Arg AlaLeu Asp Leu Ser 575 580 585 Leu Lys Tyr His Phe Val Thr Pro Leu Thr SerMet Val Val Thr 590 595 600 Lys Pro Glu Asp Asn Glu Asp Glu Arg Ala IleAla Asp Lys Pro 605 610 615 Gly Glu Asp Ala Glu Ala Thr Pro Val Ser ProAla Met Ser Tyr 620 625 630 Leu Thr Ser Tyr Gln Pro Pro Gln Asn Pro TyrTyr Tyr Val Asp 635 640 645 Gly Asp Pro His Phe Ile Ile Gln Ile Pro GluLys Asp Asp Ala 650 655 660 Leu Cys Phe Asn Ile Asp Glu Ala Pro Gly ThrVal Leu Arg Leu 665 670 675 Ile Gln Asp Ala Val Thr Gly Leu Thr Val AsnGly Gln Ile Thr 680 685 690 Gly Asp Lys Arg Gly Ser Pro Asp Ser Lys ThrArg Lys Thr Tyr 695 700 705 Phe Gly Lys Leu Gly Ile Arg Asn Ala Gln MetAsp Phe Gln Val 710 715 720 Glu Val Thr Thr Glu Lys Ile Thr Cys Gly ThrGly Arg Ala Ser 725 730 735 Thr Phe Ser Trp Leu Asp Thr Val Thr Val ThrGln Asp Gly Leu 740 745 750 Ser Met Met Ile Asn Arg Lys Asn Met Val ValSer Phe Gly Asp 755 760 765 Gly Val Thr Phe Val Val Val Leu His Gln ValTrp Lys Lys His 770 775 780 Pro Val His Arg Asp Phe Leu Gly Phe Tyr ValVal Asp Ser His 785 790 795 Arg Met Ser Ala Gln Thr His Gly Leu Leu GlyGln Phe Phe Gln 800 805 810 Pro Phe Asp Phe Lys Val Ser Asp Ile Arg ProGly Ser Asp Pro 815 820 825 Thr Lys Pro Asp Ala Thr Leu Val Val Lys AsnHis Gln Leu Ile 830 835 840 Val Thr Arg Gly Ser Gln Lys Asp Tyr Arg LysAsp Ala Ser Ile 845 850 855 Gly Thr Lys Val Val Cys Trp Phe Val His AsnAsn Gly Glu Gly 860 865 870 Leu Ile Asp Gly Val His Thr Asp Tyr Ile ValPro Asn Leu Phe 875 880 885

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) an amino acidsequence of SEQ ID NO:1, b) a naturally-occurring amino acid sequencehaving at least 90% sequence identity to the sequence of SEQ ID NO:1, c)a biologically-active fragment of the amino acid sequence of SEQ IDNO:1, and d) an immunogenic fragment of the amino acid sequence of SEQID NO:1.
 2. An isolated polypeptide of claim 1, having a sequence of SEQID NO:1.
 3. An isolated antibody which specifically binds to apolypeptide of claim
 1. 4. A diagnostic test for a condition or diseaseassociated with the expression of GAPIP in a biological samplecomprising the steps of: a) combining the biological sample with anantibody of claim 3, under conditions suitable for the antibody to bindthe polypeptide and form an antibody: polypeptide complex; and b)detecting the complex, wherein the presence of the complex correlateswith the presence of the polypeptide in the biological sample.
 5. Theantibody of claim 3, wherein the antibody is: (a) a chimeric antibody;(b) a single chain antibody; (c) a Fab fragment; (d) a F(ab′)2 fragment;or (e) a humanized antibody.
 6. A composition comprising an antibody ofclaim 3 and an acceptable excipient.
 7. A method of diagnosing acondition or disease associated with the expression of GAPIP in asubject, comprising administering to said subject an effective amount ofthe composition of claim
 6. 8. A composition of claim 6, wherein theantibody is labeled.
 9. A method of diagnosing a condition or diseaseassociated with the expression of GAPIP in a subject, comprisingadministering to said subject an effective amount of the composition ofclaim
 8. 10. A method of preparing a polyclonal antibody with thespecificity of the antibody of claim 3 comprising: a) immunizing ananimal with a polypeptide of SEQ ID NO:1 or an immunogenic fragmentthereof under conditions to elicit an antibody response; b) isolatingantibodies from said animal; and c) screening the isolated antibodieswith the polypeptide thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide of SEQ ID NO:1.
 11. An antibodyproduced by a method of claim
 10. 12. A composition comprising theantibody of claim 11 and a suitable carrier.
 13. A method of making amonoclonal antibody with the specificity of the antibody of claim 3comprising: a) immunizing an animal with a polypeptide of SEQ ID NO:1 oran immunogenic fragment thereof under conditions to elicit an antibodyresponse; b) isolating antibody producing cells from the animal; c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide of SEQ ID NO:1.
 14. Amonoclonal antibody produced by a method of claim
 13. 15. A compositioncomprising the antibody of claim 14 and a suitable carrier.
 16. Theantibody of claim 3, wherein the antibody is produced by screening a Fabexpression library.
 17. The antibody of claim 3, wherein the antibody isproduced by screening a recombinant immunoglobulin library.
 18. A methodfor detecting a polypeptide of SEQ ID NO:1 in a sample comprising thesteps of: a) incubating the antibody of claim 3 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide of SEQ ID NO:1 in the sample.19. A method of purifying a polypeptide of SEQ ID NO:1 from a sample,the method comprising: a) incubating the antibody of claim 3 with asample under conditions to allow specific binding of the antibody andthe polypeptide; and b) separating the antibody from the sample andobtaining purified polypeptide of SEQ ID NO:1.